Microfluidics-enabled multimaterial stereolithographic printing

ABSTRACT

Described are systems and methods for multi-material printing. The systems and methods can utilize a stereolithographic printing device, a moving stage, and a microfluidic device. The microfluidic device can include a plurality of reservoirs, each reservoir housing a different ink for printing, and a microfluidic chip. The microfluidic chip can include a chamber that comprises a plurality of inlets, a printing region, and one or more outlets as well as an elastic membrane.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/749,318, entitled “MICROFLUIDICS-ENABLED MULTIMATERIALSTEREOLITHOGRAPHIC BIOPRINTING,” filed Oct. 23, 2018. The entirety ofthis application is hereby incorporated by reference for all purposes.

TECHNICAL FIELD

The present disclosure relates generally to systems and methods formulti-material printing including multi-material bioprinting.

BACKGROUND

Three dimensional (3D) printing is an additive manufacturing method thatcan be used to create a wide variety of objects. In some instances, 3Dprinting can produce objects that cannot otherwise be created usingtraditional manufacturing processes. For example, in stereolithography,to create an object using a 3D printer, a layer of material isdeposited, for example, photochemically solidified across a 2D plane,and then another layer of material is deposited on top of the previouslayer. This process is repeated multiple times until the final object isobtained.

One particular type of 3D printing is 3D bioprinting where biologicalmaterials are incorporated into the inks used for printing. It has beenfound that bioprinting can be used to fabricate biomedical constructs,such as artificial tissues, tissue models, functional biomaterials,biomolecules, biomedical devices, scaffolds, and the like. 3Dbioprinting is useful because existing manufacturing methods such asfreeze-drying and salt-leaching lack flexibility to tune the designregionally.

Although strides have been made to improve 3D bioprinting, several keychallenges remain, including the continuous fabrication of cell-ladenconstructs with clinically relevant dimensions and the inability tobioprint multicomponent complex constructs with high precision.Additionally, a core challenge has involved managing material deliverywhen using multiple materials to fabricate the constructs.

SUMMARY

The present disclosure relates generally to systems and methods formulti-material printing and, more particularly, to systems and methodsfor multi-material bioprinting. Notably, the systems and methods of thepresent disclosure can utilize a stereolithographic printing device torapidly fabricate biological constructs with high precision and withclinically relevant dimensions.

In one aspect, the present disclosure can include a 3D printing system.The 3D printing system can include a stereolithographic printing device,a moving stage, and a microfluidic device where the microfluidic devicecan include a plurality of reservoirs, each reservoir housing adifferent ink for printing, and a microfluidic chip.

In another aspect, the microfluidic chip comprises a chamber, whereinthe chamber comprises a plurality of inlets, a printing region, and oneor more outlets. In one instance the chamber is comprised of apolydimethylsiloxane (PDMS) polymer.

In a further aspect, the reservoirs housing the inks can be individuallyconnected to a respective inlet present on the microfluidic chip.

In another aspect, the microfluidic chip can include an elasticmembrane. The elastic membrane can be bonded to the chamber.Furthermore, the elastic membrane can, for example, be made out of aPDMS polymer.

In a further aspect, the chamber bonded to the elastic membrane issandwiched between two polymer sheets. The two polymer sheets can be,for example, comprised of poly(methyl methacrylate) (PMMA).

In an additional aspect, the printing device can comprise a digitalmicromirror device (DMD).

In another aspect, each reservoir is connected to a pneumatic valvewherein the pneumatic valves are each connected to a containercomprising a gas.

In yet a further aspect, the inks used in the stereolithographicprinting system can include gelatin methacryloyl (GeIMA) andpoly(ethylene glycol) diacrylate (PEGDA). Additionally, the inks caninclude biologically active components such as biomaterials, cells,growth factors, cytokines, anti-infection agents, adhesive molecules,and nanoparticles.

In another aspect, the present disclosure can include a method ofprinting a multi-material 3D construct comprising providing a pluralityof inks; releasing at least one first ink into a microfluidic chipwherein the microfluidic chip comprises (i) a chamber, wherein thechamber further comprises a printing region for holding the ink to beprinted; (ii) a deposition layer, and (iii) an elastic membrane; causingthe elastic membrane to deform; photocrosslinking the first ink in theprinting region onto the deposition layer to form a first printed layer;reducing the deformation of the elastic membrane; washing the chamber;and releasing at least one second ink into the microfluidic chip toproduce a second printed layer.

In a further aspect, the chamber is washed using the second ink. Inanother aspect, the chamber is washed using a buffer.

In yet another aspect, one of the first or second inks can comprise abiologically active component, wherein the biological active componentis selected from biomaterials, cells, growth factors, cytokines,anti-infection agents, adhesive molecules, and nanoparticles.

In another aspect, the first and second inks can both comprise abiologically active component, wherein the biological active componentis selected from biomaterials, cells, growth factors, cytokines,anti-infection agents, adhesive molecules, and nanoparticles.

In yet another aspect, two or more first inks are released into themicrofluidic chip

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present disclosure will becomeapparent to those skilled in the art to which the present disclosurerelates upon reading the following description with reference to theaccompanying drawings, in which:

FIG. 1 is a block diagram illustrating an example of a system formulti-material printing according to an aspect of the presentdisclosure;

FIG. 2 is a block diagram illustrating an example of an injection systemthat can be used in connection with the system of FIG. 1 ;

FIG. 3 is a block diagram illustrating an example of astereolithographic printing device that can be used in connection withthe system of FIG. 1 ;

FIG. 4 is a block diagram illustrating an example of microfluidic chipthat can be used in connection with the system of FIG. 1 ;

FIG. 5 is a process flow diagram of an example method for multi-materialprinting according to another aspect of the present disclosure;

FIGS. 6 a-6 c provides schematics showing the setup of a system formulti-material bioprinting; (a) provides planar schematics showing thesetup of the bioprinter, including an ultraviolet (UV) lamp (385 nm),optical lenses and objectives, a DMD chip, and a microfluidic device;(b) provides a schematic showing the actual setup of the entire opticalplatform; (c) provides a schematic showing an open-chamber microfluidicchamber used to create single-material printouts.

FIGS. 7 a-7 f provides examples of PEGDA-50% constructs printed by theopen-chamber setup; (a) shows the role of UV exposure time on regulatingthe bioprinting resolution: oblique lines printed for differentUV-exposure durations; (b) and (c) show a 2D resolution assessment byprinting tree-like structures; (d) provides a bioprinted sample 3Dconstruct; (e) provides a bioprinted two-material construct made bymanually washing the first ink and adding the second ink; (f) shows aresolution map by printing thin lines at an UV intensity of 100 mW cm⁻².

FIGS. 8 a-8 f provides images regarding the microfluidic chip; (a) is aschematic showing the assembly of the microfluidic chip having fourinlets and one common outlet; (b) shows the operation of themicrofluidic device for consecutive injection of different bioinks andthe washes in between the injections; (c) shows the defined computerfluid dynamics (CFD) model and the velocity profile (m/s) of PEGDA (witha density 1.06 kg/m³ and a viscosity 1×10⁻⁵ Pa s) in the closed chamberunder sinusoidal fluid flow; (d)-(f) show the role of mixing and washingobserved by flow streamlines in GeIMA solution (15% w/v) mixed by fooddye in the microfluidic chip for a star pattern, two rectangularpatterns (made of PMMA molds), and no pattern, respectively;

FIGS. 9 a-9 c provides images and data regarding the star shape in themicrofluidic chip; (a) shows a comparison of experimental flow contours(left) and CFD result for velocity contours (right) around a star shapein the microfluidic chamber; (b) shows maximum stress and velocityvalues around the star shape versus inlet pressure and velocity in thefirst channel. (c) shows maximum stress values versus both inletvelocity and fluid viscosity.

FIGS. 10 a-10 g shows one way in which the printing system can operate;(a) is a schematic showing the microfluidic chip containing a movingpart at the center of the bottom chamber; (b) shows the computationaldomain of the finite element analysis built for an isotropic,incompressible, hyperelastic membrane supported on the rigid piston; (c)provides a simulation result showing principle strain and stress valuesof the PDMS membrane at 4-mm displacement; (d) provides schematicsshowing the four-step bioprinting process inside the microfluidic chipfor fabricating 3D objects; (e)-(g) show examples of multi-componentbioprinted constructs: (e) a two-component GeIMA-7% construct filled byfluorescent dyes (left) and a three-component pattern of coloredPEGDA-50% (right); (f) a 3D fluidic mixer made by three different colorsprinted from PEGDA-50%; and (g) a single-component and a three-componentstar-shaped pyramid of PEGDA-50%;

FIGS. 11 a-11 c provides graphs regarding the PSMA membrane; (a)provides a computational domain of the finite element analysis built forthe PDMS membrane; (b) shows experimental and numerical histories ofnormal load (applied by the piston on the PDMS membrane) anddisplacement of the piston; (c) shows the numerical estimation ofmaximum strain that occurred in the PDMS membrane for two differentthicknesses.

FIGS. 12 a-12 c provides examples of cell patterning; (a) shows a musclestripe-like shape: C2C12 skeletal muscle cells-loaded GeIMA-7%immediately after bioprinting; (b) shows a star shape: mesenchymal stemcells (MSCs) printed in a PEGDA-50% pattern after 24 h; (c) shows areticular network made by osteoblast-loaded GeIMA-7% immediately afterbioprinting. The designed mask is shown for each case.

FIGS. 13 a-13 c shows schematics and corresponding printed constructs;(a) shows a tumor angiogenesis model: i) schematic showing the tumorangiogenesis; ii) schematic of the mask for printing; iii) bioprintedmicrovasculature in PEGDA; iv) bioprinted MCF7 breast tumor cells-ladenmicrovascular bed of GeIMA further seeded with HUVECs in the channels;(b) shows a skeletal muscle model: i) schematic showing the skeletalmuscle tissue; ii) schematic of the mask for printing; iii) bioprintedstructure of GeIMA containing patterned C2C12 cells and fibroblastsafter 48 h of culture; iv) Presto Blue measurements of cellproliferation in the bioprinted structures; (c) shows a tendon-to-boneinsertion model: i) schematic of the tendon-to-bone insertion site; ii)schematic of the mask for printing; iii) bright-field optical micrographshowing a bioprinted dye-laden GeIMA structure; iv) bioprinted structureof GeIMA containing patterned osteoblasts, MSCs, and fibroblasts.

FIGS. 14 a-14 b shows the effects of washing on the cell viability ofbioprinted NIH/3T3 fibroblast-laden GeIMA-7% patterns; (a) showssingle-component parallel lines without washing; (b) shows two-componentcrossing networks with washing.

FIGS. 15 a-15 d provides images of a construct containing vascularendothelial growth factor (VEGF); (a) provides a concentration-gradientmodel generated by multi-material DMD bioprinting: i) schematic of theconstruct showing the PEGDA (35% v/v) frame and three GeIMA strips of 5,10, and 15% w/v mass concentrations with a uniform thickness of 1 mm;ii) a bioprinted model where the GeIMA strips contained fluorescentbeads; iii) the rat subcutaneous model used to assess the bioprintedconstructs; (b) provides photographs showing the retrieved implants atDay 10 and Day 30, along with confocal images of the retrievedconstructs at Day 30 stained for nuclei and for CD31; (c) showsimmunostaining of the retrieved implants for CD31, for different GeIMAconcentrations (5, 10, and 15%), in the absence and presence of VEGF;the nuclei were counterstained with 4′,6-diamidino-2-phenylindole(DAPI); (d) shows H&E staining of the retrieved implants for differentGeIMA concentrations, in the absence and presence of VEGF.

DETAILED DESCRIPTION Definitions

In the context of the present disclosure, the singular forms “a,” “an”and “the” can also include the plural forms, unless the context clearlyindicates otherwise.

The terms “comprises” and/or “comprising,” as used herein, can specifythe presence of stated features, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, steps, operations, elements, components, and/or groups.

As used herein, the term “and/or” can include any and all combinationsof one or more of the associated listed items.

Additionally, although the terms “first,” “second,” etc. may be usedherein to describe various elements, these elements should not belimited by these terms. These terms are only used to distinguish oneelement from another. Thus, a “first” element discussed below could alsobe termed a “second” element without departing from the teachings of thepresent disclosure. The sequence of operations (or acts/steps) is notlimited to the order presented in the claims or figures unlessspecifically indicated otherwise.

As used herein, the term “bioprinting” can refer to a method ofbiofabrication using a printer to print materials incorporated withliving cells or structures that are used for culturing cells or fortissue interactions. Bioprinting can include two-dimensional (2D)bioprinting and three-dimensional (3D) bioprinting.

As used herein, the term “2D bioprinting” can refer to a particularmeans of fabrication of planar biomedical constructs. For example, 2Dbioprinting can refer to the deposition of a single layer ofmicrodroplets or photochemically solidifying material to create a planarbiomedical construct.

As used herein, the term “3D bioprinting” can refer to a particularmeans of fabrication of 3D biomedical constructs. As an example, 3Dbioprinting can refer to particularly processes where successive layersor rows of microdroplets or material are deposited or photochemicallysolidified under computer control to create the 3D biomedical construct.As an example, a 3D biomedical construct can include a complexbiological structure comprising one or more independentthree-dimensional constructs.

As used herein, the term “biomedical construct” can refer to acombination of one or more bioprinted materials that incorporate visibleliving cells. Examples of biomedical constructs include artificialtissues, tissue models, functional biomaterials, biomolecules,biomedical devices (e.g., including multiple components likebioelectronics and high-throughput point-of-care devices), scaffolds,and the like. In some instances, biomedical constructs can be planar 2Dstructures fabricated via 2D bioprinting. In other instances, biomedicalconstructs can be 3D structures fabricated via 3D bioprinting.

As used herein, the term “bioink” can refer to a fluid, solid, orhydrogel deposited by a bioprinter. The composition of the bioink caninclude one or more biological active components, like biomaterials,cells, growth factors, cytokines, anti-infection agents, adhesivemolecules, nanoparticles, or the like.

As used herein, the terms “subject” and “patient” can be usedinterchangeably to refer to any warm-blooded living organism including,but not limited to, a human being, a pig, a rat, a mouse, a dog, a cat,a goat, a sheep, a horse, a monkey, an ape, a rabbit, a cow, etc.

Overview

The present disclosure relates generally to multi-material printing. Theuse of multiple inks to print 3D constructs has traditionally beenproblematic due to the time and labor it takes to switch between inksand to decontaminate the system. Additionally, with specific regard tobioprinting, traditionally employed techniques have numerousdisadvantages. The traditional techniques do not allow for the precisefabrication of multicomponent constructs. The instant printer systemovercomes these disadvantages. It has been found that integrating amicrofluidic device into the design of a stereolithographic printingdevice allows for an automated multi-material stereolithographicprinting system that is fast and provides constructs at high fidelity.In some instances, the multi-material printer system can be used togenerate constructs for biological in vivo use.

Systems

FIG. 1 illustrates a system 100 for multi-material printing in which aplurality of inks can be delivered in a continuous manner for rapidfabrication of constructs, including biological constructs. The system100 can include a microfluidic device 102. In one example, themicrofluidic device can include an injection system 104, a plurality ofreservoirs 106 (R1-RM, where M is an integer greater than 1), and amicrofluidic chip 108. The injection system 104 can be connected to thereservoirs 106.

Exemplary injection systems 104 include those based on variousmechanisms including a pneumatic pressure change, a mechanical pressurechange, a thermal activation, and a piezo electric activation. In someinstances a combination of mechanisms may be used. In one specificexample, the injection system can be based on a pneumatic pressurechange. FIG. 2 illustrates an injection system that includes a gassource 110 and a plurality of pneumatic values 112 (P1-PM, where M is aninteger greater than 1), where the pneumatic values are individuallyconnected to reservoirs 106.

Each of the plurality of reservoirs 106 shown in FIG. 1 can include anink for printing. In some instances, each of the reservoirs can includea different and unique ink. In other instances, two or more of thereservoirs can include the same ink. The ink may be comprised of anymaterial that is able to be photocrosslinked. Exemplary inks includehydrogel inks such as GeIMA and PEGDA. Additionally, commerciallyavailable resins can be used. In some instances the ink can include oneor more biologically active components. The biological active componentscan include biomaterials, cells, growth factors, cytokines,anti-infection agents, adhesive molecules, and nanoparticles. In someinstances, dyes and fluorescence beads may be included in the inks toassist in visualization. Additionally, in some instances,photo-absorbers can be included in the inks.

In further instances, one or more of the reservoirs 106 can contain awashing solution. For example, the washing solution may be a buffersolution such as a phosphate-buffered saline solution. A washingsolution can be used when a previously used ink is hard to remove fromthe system. In instances when the ink is not difficult to remove thesystem, the inks may be used sequentially. In this instance the laterused ink can wash out the previously used ink from the system.

The reservoirs 106 can be connected to a microfluidic chip 108.Microfluidic chip 108 will be discussed in greater detail below withregard to FIG. 4 .

Microfluidic chip 108 can be positioned on a moving stage 114. Themoving stage 114 can move in three directions (e.g., along the x, y, andz axes).

The system 100 can also include a stereolithographic printer (SLP)device 116. Exemplary SLP devices include a digital light processingdevice (DLP) and a digital micromirror device (DMD). DMD-basedprojection printing has emerged as a high-throughput DLP techniqueoffering biocompatibility for cell seeding and encapsulation. DMD is amicro-electro-mechanical system that enables a user to control over onemillion small mirrors to turn-on or turn-off on the order of kHz. An UVlamp projects light on the DMD panel, in which patterns the image ofeach layer of the computer-aided-design (CAD) model, and projects intothe bottom side of the container. Following this UV exposure, thephotosensitive polymer or hydrogel crosslinks and attaches to theprevious layer. DMD-based printing offers high-quality surface finishingand a variety of material options.

In the instance that the SLP device 116 includes a DMD, FIG. 3 showsthat the DMD can include a DMD panel 118 and a light source 120. In someinstances the light source 120 is a UV source or a visible light source.In certain instances the light source is an LED source. Additionally, insome instances one or more lens' 122 may be used to affect the focallength or resolution of the constructs being printed. In one instance,the light source can be directed via an optical path toward the DMDpanel at specific angles to facilitate light reflection to the stagethrough the lens.

The SLP device 116, the moving stage 114, and the microfluidic device102 can be coupled to a controller 124. In some instances the controller124 can be a master controller coupled to one or more subcontrollers. Inone instance the controller 124 may include a CAD and computer-aidedmanufacturing (CAD/CAM) system to, for example, design the products tobe created or convert images to a product design, control the reservoirs106, control the moving stage 114, and control the SLP device 116. SuchCAD/CAM systems are known to those skilled in the art and can beimplemented in combination with the present disclosure in accordancewith known techniques or variations thereof that will be apparent tothose skilled in the art.

FIG. 4 illustrates a microfluidic chip 108. Beneath the microfluidicchip 108 a tower 202 can be present.

The microfluidic chip 108 can be comprised of multiple layers. Forexample, the microfluidic chip 108 may be comprised of five layers,including two outer layers 204 and 212, a deposition layer 206, achamber 208, and an elastic membrane 210. As seen in FIG. 2 , outerlayer 212 can have a section removed so that the tower 202 can fitthrough the removed section. In some cases, the microfluidic chip 108can be comprised of three layers, including a deposition layer 206, achamber 208, and an elastic membrane 210.

Chamber 208 can include a plurality of inlets 218 through which thevarious inks can flow. For example, there may be two, three, four, five,or six inlets. The plurality of inlets allows for sequential injectionof different inks. The chamber 208 can also include a printing region214. The printing region is the section of the chamber where the ink tobe photocrosslinked and printed resides. Additionally, the chamber canhave one or more outlets 216 through which the ink being washed out ofthe system can be discarded, recirculated, or reused.

The elastic membrane 210 can be bonded to chamber 208 through, forexample, chemical bonding or mechanical force. The elastic membrane 210can function to seal the chamber 208. For example, during the printingprocess ink can be ejected from reservoir 106 into the chamber 208 andinto printing region 214. In one instance, the microfluidic chip 108 canthen be lowered such that the tower 202 comes in contact with theelastic membrane 210. This contact can result in the deformation of theelastic membrane, and the degree of contact can provide the designatedthickness for the layer to be photocrosslinked. The ink in the printingregion 216 can then be photocrosslinked. After the ink has beenphotocrosslinked, the microfluidic chip 108 can be raised which canreduce the deformation of the elastic membrane 210. The ink can then bewashed from the system. In another instance, when the ink is ejectedfrom reservoir 106 into chamber 208, the tower 202 can already be at anelevated position and the elastic membrane 210 can already be deformedto the level needed to achieve the designated thickness of thephotocrosslinked layer. The ink can then be photocrosslinked, and thetower 202 can be lowered which can reduce the deformation of the elasticmembrane 210. The ink can then be washed from the system.

Tower 202 can be made out of any optically transparent material. Forexample, tower 202 can be made of glass, plastic, or elastomer that isoptically transparent. In one instance, tower 202 can be made of apolymer such as PMMA, PDMS, or polystyrene. In one instance, the tower202 is hollow. In another instance, the tower 202 is not hollow.

Outer layers 204 and 212, deposition layer 206, and chamber 208 can bemade out of any suitable material, such as glass, plastic, or elastomer.In one instance, the outer layers 204 and 212 can be polymer sheets. Thepolymer can be, for example, PMMA, PDMS, or polystyrene. In oneparticular example, the outer layers 204 and 212 can be PMMA sheets. Inanother instance, the deposition layer 206 can be a glass sheet. In afurther instance, chamber 208 can be a PDMS sheet.

The elastic membrane 210 can be made out of an optically transparentflexible material. In one instance the elastic membrane 210 material isoxygen permeable. In certain instances the elastic membrane 210 can bemade out of a polymer such as polybutylene adipate terephthalate (PBAT)or PDMS. The elastic membrane can also be made of a plastic such asthermoplastic polyurethane or Teflon® AF 2400. In one particularinstance, the elastic membrane 210 is a PDMS membrane.

In another aspect, a mixer can be used to mix two or more inks together.In one instance the mixer can be included in microfluidic chip 108. Forexample, the mixer may be placed in chamber 208 prior to reachingprinting region 214. In another instance, the mixer can be placedbetween reservoirs 106 and microfluidic chip 108. The mixer can be amicrochannel mixing device. For example, the mixer may be designed inthe manner disclosed by Stroock et al., Chaotic Mixer for Microchannels,Science, 295:647-51 (Jan. 25, 2002) where patterned groves on the floorof a channel are utilized.

One skilled in the art would understand that other configurations of thesystem 100 can be used to print 3D constructs. For example, the printedconstruct can be fabricated using a bottom up approach where themicrofluidic chip 108 is positioned below the SLP device 116.

Methods

Another aspect of the present disclosure can include a method 300 (FIG.5 ) for multi-material printing. The multi-material printing can beaccomplished, for example, by the system 100 of FIG. 1 . The method 300of FIG. 5 is illustrated as a process flow diagram. For purposes ofsimplicity, the method 300 is shown and described as being executedserially; however, it is to be understood and appreciated that thepresent disclosure is not limited by the illustrated order as some stepscould occur in different orders and/or concurrently with other stepsshown and described herein. Moreover, not all illustrated aspects may berequired to implement the method 300.

FIG. 5 illustrates a method 300 for multi-material printing. At 302, aplurality of inks can be provided to reservoirs 106. At 304, a first inkcan then be released from reservoir 106 into the microfluidic chip 108using the injection system 104. In some instances more than one ink canbe released at a time. In even further instances, when more than one inkis released, the inks can be mixed before or after they arrive atmicrofluidic chip 108. Within microfluidic 108, the ink to be printedcan be held in the printing region 214 of chamber 208. Chamber 208 canhave an elastic membrane 210 bound to it.

At 306, the elastic membrane 210 can become deformed. For example,microfluidic chip 108 can be lowered such that the tower 202 comes incontact with the elastic membrane 210. This contact can result in thedeformation of the elastic membrane and the degree of contact canprovide the designated thickness for the layer to be photocrosslinked.In another example, the tower 202 can be raised such that it comes intocontact with the elastic membrane 210 causing the elastic membrane 210to deform. The degree of contact between the tower 202 and elasticmembrane 210 can provide the designated thickness for the layer to bephotocrosslinked. In some instances, the tower 202 can already be raisedand the elastic membrane 210 can already be deformed before the ink isreleased into the printing region 214 of microfluidic chip 108.

At 308, the ink in the printing region 214 can then be photocrosslinkedusing the SLP device 116. The photocrosslinked ink can be deposited ontothe deposition layer 206 to form a first printed layer.

At 310, after the ink has been photocrosslinked, the microfluidic chip108 can be raised which can reduce the deformation of the elasticmembrane 210. The degree that the microfluidic chip 108 is raised can bebased on the amount of space that is needed to efficiently remove theink from the system. In another instance, the tower 202 can be loweredwhich can reduce the deformation of the elastic membrane 210. The degreethat the tower 202 is lowered can be based on the amount of space thatis needed to efficiently remove the ink from the system. In even furtherinstances, the position of the microfluidic chip 108 and tower 202 canremain the same.

At 313, the ink can then be washed from the system including chamber208. In one instance, the chamber 208 is washed using the next ink to beprinted. In another instance, the chamber 208 is washed using a buffer.

At 314, a second ink can be released from reservoir 106 into themicrofluidic chip 108 to produce a second printed layer. In certaininstances, more than one second ink can be released from reservoirs 106into the microfluidic chip 108. In instances when more than one ink isreleased, the inks can be mixed before or after they arrive atmicrofluidic chip 108.

The process starting at either 302 or 304 can then be repeated until theconstruct is fabricated.

The first or second inks can comprise one or more biologically activecomponents. Exemplary biologically active components includebiomaterials, cells, growth factors, cytokines, anti-infection agents,adhesive molecules, and nanoparticles.

The 3D printers described herein can be used to in a variety ofapplications including tissue engineering, regenerative medicine, andbiosensing. For example, the 3D printers described herein can be used toprepare musculoskeletal systems, to prepare biomimetic cancer models,and to prepare implants for in vivo use.

In one particular instance the 3D printers can be used to prepareimplants that stimulate angiogenesis in vivo. The method can includepreparing an implant using the method described above where one or moreof the inks includes VEGF. The implant can then be inserted into thebody of a subject and the ability of the implant to stimulateangiogenesis can be monitored.

Experimental

The following example is shown for the purpose of illustration only andis not intended to limit the scope of the appended claims. This exampleillustrates that the integration of a simple microfluidic platform canadvance the DMD-based bioprinter for proper fabrication of inhomogeneousconstructs at high fidelity. The microfluidic platform allowed forintegration of multiple independent bioink injections and furtheroffered easy feeding of different materials with fast switching.Computational fluid dynamics was used to assess the performance of themicrofluidic system for multi-material patterning. Various patterns werefabricated through this platform to validate its multi-materialbioprinting capability. The flexibility and biocompatibility of theplatform to generate biomimetic heterogeneous tissue constructs wasfurther evaluated by using bioinks loaded with multiple cell types,introduced from the microfluidic chips into the DMD bioprinter.

Bioprinting System

The DMD-based bioprinter uses UV light (up to 500 mW/cm²) to polymerizea liquid pre-polymer towards a solid structure (FIG. 6 ). The DMD panelthat is an array of reflective-coated micro-mirrors creates lightpatterns at high definition (i.e. 1050×920) and speed (10 kHz rate ofswitching). The digital state of each micro-mirror can be controlled asbeing either 0 (dark) or 1 (light-reflecting for photo-polymerization)while the bioink is introduced to the focal plane of the projectedimage, leading to its crosslinking in a layer-by-layer fashion. Theimage size was calibrated by printing a single image featuring a gridpattern, and then measuring the grid by a light microscope. The lateralresolution is theoretically limited by the physical size of DMD mirrors,which is 7.6 μm for the selected model; however, experimental printingresolution (i.e., smallest feature size) was determined at the order of10 μm (FIG. 7 ). Simple patterns were used to show printing capabilitiesof our DMD-based bioprinter over a range of UV exposure parameters andphotoinitiator concentrations. The photoinitiator concentration affectedthe time required to fully crosslink the hydrogel from seconds tominutes. The practical resolution of our bioprinter was further shown byprinting parallel lines, in which it generated a line thickness down to˜25 μm (FIG. 7 f ).

Different from existing DMD bioprinters, a unique microfluidic devicewas developed to turn the system into a multi-materialstereolithographic bioprinting platform. FIG. 8 illustrates the designof the microfluidic device consisting of one PDMS chamber held betweentwo PMMA sheets and four inlets, which allowed for sequential injectionof different bioinks (FIG. 8 b ). The three branches in the middleregion were introduced to widen the directionality of the washing flowand reduce flow forces imposed on the printed construct (as seen byexperimental observations in FIG. 8 ). Bioink flow filled the chip in afew seconds, before subjecting to UV crosslinking. Phosphate-bufferedsaline (PBS) then washed away the first bioink within the same timeframe, and this was repeated for the second and following bioinks. Inaddition, chip performance was assessed by tracking dye particles indye-filled bioink flows within the microfluidic device and washing bythe subsequent bioink (in a different color), or PBS. FIG. 8 c shows thesimulation of our numerical model for flow patterns around the starshape, indicating a close similarity (correlation coefficient >0.70) inthe flow patterns observed around the star shape (FIG. 9 ); left imageversus right image). FIGS. 8 d-f provide a direct visual impression onhow the bioinks were washed when different shapes were printed insidethe chamber. Despite the presence of branching and potential turbulenceat edges, reasonable laminar flow in the printing region was able to beobserved with a high-speed digital camera. As expected formicro-channels, the viscous bioink was reaching to the printing regionin a laminar regime (Re˜10-100), which allowed smooth transition betweensequential bioink injections. However, the shape of printed constructrestricted the velocity and duration of bioink feeding. The bioink couldbe easily washed by the subsequent flow in the case of straight lines(FIG. 8 e ), in the absence of any dead zone or low-speed streamlines inthe flow (e.g., washing time ˜5 s at an inlet velocity of 1 cm s⁻¹). Incontrast, flow patterns in the case of star shape showed low-speedstreamlines inside the cavities that hampered the washing process, thusrequiring bioink flow at higher speed or longer time (e.g., washing time˜20 s at an inlet velocity of 1 cm s⁻¹). When there was no objectpresent in the chamber the flow pattern was smooth (FIG. 8 f ); e.g.,washing time ˜2 s at an inlet velocity of 1 cm s⁻¹). Moreover, it wasinvestigated how the inlet pressure, regulated by nitrogen tank, and thebioink viscosity could control maximum stress applied on the printed gelwith a star shape (selected as a standard here). As summarized in FIG. 9b-c , both stress and fluid velocity values are linearly correlated byinlet pressure or inlet velocity. This may allow the prevention of geldisplacement by high shear stresses.

An elastomeric membrane made of PDMS was built into the microfluidicchip. The elastomeric membrane undergoes deformation during thebioprinting process to allow for the construction of 3D objects inconjunction with programmed injection of bioinks (FIG. 10 a-b ).Starting from the first layer of crosslinking, the microfluidic chipmoves up and this movement yields reduced deformation on the membrane,until its resting position (i.e., for a 5-mm-high printed construct).The resilience of the membrane was studied through numerical simulationand customized mechanical testing, as summarized in FIGS. 10 c and 11.The hyperelastic Neo-Hookean model^([19]) was used to simulate thedeformation field and stress distribution of the PDMS membrane for theimposed boundary conditions (FIG. 11 ). The maximum deformation wasunder 50% strain when the chip was set at the initial position and itoccurred at the contact region between the membrane and the rigid shellfor the range of membrane thickness (from 200 μm to 500 μm). The centralregions had uniform strains lower than 50%, thus the deformation fieldis still within the elastic (i.e., reversible) range of PDMS. Uniaxialtester was then employed to obtain load-displacement history of themembrane under vertical movements of the rigid shell (FIG. 11 ), whereload-displacement curves showed hysteresis response depicting the roleof friction forces on the membrane function. The membrane was programmedto load-unload when the chip was filled for printing each layer andsubsequent washing, as depicted in FIG. 10 d ; however, it was foundthat the presence of friction forces that yielded residual strain to acertain degree limited the number of cycles for membrane deformation,requiring future optimization for printing a very high number of layers.The membrane was manually tightened after each construct when arelatively small thickness was printed and removed from the microfluidicchip. To the end, for printing large numbers of layers in a singleconstruct such residual strain may become an issue requiring furtheroptimizations. It should be noted that the elastomeric membranefunctions to seal the microfluidic chamber during the exchange of thedifferent bioinks while providing the ability of 3D bioprinting throughlayer-by-layer photocrosslinking. In comparison, in a conventional DMDor DLP setup, the open-chamber design would not allow for efficientinjection or washing of the bioinks, and is therefore prohibitive ofmulti-material stereolithographic bioprinting.

The performance of the membrane was validated using a numerical model.The thickness of the PDMS membrane was in a range of 200-500 μm and itwas subjected to movements of the tubular piston in a range of 0-4 mm.The elasticity of the PDMS membrane was obtained using Instron tensiletester (Instron, model 3300) and fit with an incompressible Neo-Hookeanmodel. The simulation was computed using FEA software (Abaqus-version6.10, HBK, Pawtucket, R. I.). FIG. 10 b demonstrates the boundaryconditions applied for numerical modeling of the membrane deformation inaxisymmetric coordinate system. The upper surface of the membrane wassupported by a rigid piston while a tubular piston was pushing themembrane from the bottom. To mesh the membrane geometry, quadrilateralaxisymmetric hybrid elements (CAX4H element type) were employed from theAbaqus/Standard library. The large displacement theory was used forformulations where the role of nonlinear effects of the largedisplacements in membrane elements were included. Full Newton approachtogether with a symmetric solver were utilized to solve the finiteelements equations. The results are summarized in FIG. 10 and FIG. 11 .

The capability of the bioprinter in generating two-dimensional (2D) and3D constructs was demonstrated. Simple shapes with different materialswere bioprinted using PEGDA (50% v/v) and GeIMA (7% w/v) solutions,containing 2, 3, and 4 colored bioinks (FIG. 10 e-g ). Constructs couldalso be fabricated in different shapes, such as eccentric circles,parallel and oblique stripes, and pyramids of different base-shapes (seealso FIGS. 7 and 12 ). The planar printing resolution demonstrated thecapacity of our bioprinting platform, while 3D bioprinting resolutionwas hampered by UV light scattering. It is noted that, while differentbioinks were sequentially injected into the microfluidic chip, there wasno obvious sign of mixing, indicating successful washing of bioinksprior to crosslinking, indicating the good performance of theelastomeric membrane in the microfluidic chip.

A set of sophisticated structures were further designed and bioprintedthat resembled biological tissues such as tumor angiogenesis, musclestrips, and musculoskeletal junctions (FIGS. 13 ai, bi, and ci). GeIMAwas used as a bioink due to its intrinsic cell adhesion moieties thatpromote cell spreading and functionality. Each organ-like structure had2-4 different bioinks as each bioink was individually patterned in arapid fashion, with smooth transitions among different bioinks. Thistransition that requires washing of bioink residuals in the bioprintingprocess can hamper cell viability as highlighted by a comparison of twodifferent cases: single-component and two-component reticular constructsin FIG. 14 .

The bioprinted structures possessed explicitly separated borders amongdifferent cell-laden bioinks, confirming the role of washing (FIG. 13 ).The printing resolution was determined to be approximately 20-30 μm,slightly reduced in comparison with non-cellular patterns due toscattering of photons from cellular components. Nevertheless, such aresolution is still compatible with our previous reports, suggestingthat the addition of the microfluidic chip in the current system tointroduce multiple bioinks did not exert noticeable effects on the DMDstereolithographic bioprinting process.

One of the current challenges in cancer biology is to understand thecomplex, multi-cellular cancer microenvironment. In vitro tumor culturescurrently used in cancer research often result in different cell-matrixassociations that in turn affect their functions; to this end,bioprinting could become a promising strategy to engineering biomimeticcancer models due to its versatility in depositing cells and matrices inprecisely defined manners. Specifically, a pattern mimickingangiogenesis in a matrix of GeIMA laden with scattered breast cancercells (MCF7) was printed, followed by introduction of human umbilicalvascular endothelial cells (HUVECs) within the vascular channels, asshown in FIG. 13 a . Such a model, although primitive, could potentiallyallow studying the tumor progression and angiogenesis.

Models of the musculoskeletal systems were also fabricated using themulti-material DMD-based bioprinter. Muscle bundle-like constructs wereprinted using two bioinks, loaded with NIH/3T3 fibroblasts and C2C12skeletal muscle cells. The fluorescence micrograph clearly revealed thecapability of the system to print the spatially distributed cell-ladenbioinks (FIG. 13 bii and iii), laying down the basis for futurefabrication of functional muscular tissues containing hierarchicalassembly of multiple cell types. The cell viability, determinedimmediately and at 1 and 7 days post-bioprinting, indicated that allcell types maintained satisfactory proliferation and metabolic activity(FIG. 13 biii). In addition, a construct mimicking the musculoskeletalinterface integrating three different cell types, MSCs, fibroblasts, andosteoblasts was printed (FIG. 13 c ). Again, the printed patterns werewell-defined showing relatively strong similarity with the model (FIG.13 ciii and iv versus ii).

Human umbilical vein endothelial cells (HUVECs, Lonza, Portsmouth, N.H.)and MSCs (Lonza) were cultured in endothelial growth medium (EGM, Lonza)and high-glucose Dulbecco's Modified Eagle Medium (DMEM; ThermoFisher,Waltham, Mass.), respectively. Human dermal fibroblasts, NIH/3T3fibroblasts, MCF7 breast cancer cells and C2C12 skeletal muscle cellswere obtained from ATCC (Manassas, Va.) and maintained in DMEM. Allmedia were supplemented with 10% fetal bovine serum (FBS, ThermoFisher,Waltham, Mass.) and 1% penicillin-streptomycin (ThermoFisher). All cellswere cultured at 37° C. with 5% CO2 and passaged prior to reachingconfluence.

The multi-material capacity of the bioprinting platform was furtherassessed in vivo in a rat subcutaneous implantation model, similar topublished protocols and as discussed below. (Oh et al., J. Biomater.Sci., Polym. Ed., 2012, 23, 2185). A four-material construct wasdesigned made of PEGDA (35% v/v) as the framing structure and threeGeIMA strips with mass concentrations of 5, 10, and 15% w/v,respectively, as presented in FIG. 15 ai. Construct with vascular VEGFloaded in the GeIMA strips was used as a positive control to stimulateangiogenesis (FIG. 15 aii and iii), where blank GeIMA hydrogels servedas the negative control. The implants were harvested at Days 10 and 30for histological examination and their gross appearances were firstassessed (FIG. 15 aiv). It has been previously reported that VEGFinduces migration of multiple endothelial cell lines, such as capillaryendothelium. As depicted in FIG. 15 b , the presence of VEGF in thebioprinted multi-material constructs did lead to the formation of moreblood vessels in the implants when compared to those without VEGF. Theexpressions of CD31 by the invaded cells were higher in the VEGFimplants at Day 10 compared to Day 30 (FIG. 15 c ). These results showedthat immobilized VEGF promoted the formation of the blood vessel networkin the bioactive GeIMA hydrogels, while the inert PEGDA served as theframe in the bioprinted multi-material structure.

In addition, VEGF seemed to have induced more pronounced inward growthof the connective tissues along the peripheries of the implants (FIG. 15b ). The hematoxylin and eosin (H&E)-staining results furtherdemonstrated the inflammatory responses of the host to the implants andrecruitment of inflammatory cells, particularly at the interfaces ofconnective tissues and implants (FIG. 15 d ). The differences in CD31expression and connective-tissue formation were less distinctive betweenthe 5% GeIMA and the 10% GeIMA compared to those in the 15% GeIMA (FIG.15 c-d ). This may indicate that the higher concentration of GeIMA (15%)prevented the invasion of cells into the implant possibly due to thedenser polymer network and reduced rate of biodegradation. The in vivostudy suggested the ability to fabricate heterogeneous constructs usingthe novel multi-material stereolithographic bioprinting strategy toregulate desired biological functions such as angiogenesis.

Animal Protocol

To test the angiogenic efficacy of the bioprinted multi-materialconstructs, hydrogel specimens were subcutaneously implanted in thedorsal region of 300-g male Wistar rats (Charles River Laboratories,Worcester, Mass.). All animal experiments were conducted according tothe NIH Guidelines for the Care and Use of Laboratory Animals. Protocolwas approved by the Institutional Animal Care and Use Committee ofBrigham and Women's Hospital (#2017N000114). The rats were divided into2 groups, the first group (n=6) was used as a control where hydrogelconstructs without VEGF were implanted; a second group (n=6) wasimplanted with the multi-material constructs with increasing massconcentrations of GeIMA. Before the implantation of the constructs,isoflurane inhalation (2.0-2.5% in air v/v) was used for anesthesia.After standardized aseptic animal preparation, a dorsal skin incision (2cm) was performed to expose the subcutaneous tissue of the rat, and 3subcutaneous pockets were created in each flank (a total of 6subcutaneous pockets), and 1 hydrogel construct (5 mm×5 mm×1 mm in size)per subcutaneous pocket was implanted. After implantation, the incisionwas closed with prolene sutures (Ethicon, Somerville, N.J.) and theanimals were returned to their respective cages for recovery. At 10 and30 days of implantation, 3 animals of each group were sacrificed underanesthesia overdose and the samples were extracted for further analysis.They were fixed in 10% neutral buffered formalin overnight and thenserially dehydrated in ethanol (10%, 30%, 50%, 70%, 90%, and 100%). Thedehydrated samples were embedded in paraffin for 48 h and sectioned in12-μm thickness. Histology (H&E) and immunostaining against CD31(ab182981, Abcam, Cambridge, Mass.) were performed to characterize cellinfiltration (inflammation), tissue remodeling, and angiogenesis. Thestained samples were imaged by an optical microscope (Zeiss, Oberkochen,Germany) and by a Leica SP5 X inverted confocal microscope (Leica,Wetzlar, Germany).

In summary, an innovative strategy has been demonstrated by integratinga microfluidic device into the design of a DMD-based bioprinter toachieve, for the first time, automated, multi-materialstereolithographic bioprinting. In a typical process, the DMD-basedbioprinting platform disclosed in this example requires only a fewseconds to preform washing (if switching is required); for example,non-uniform constructs were printed, composed of 2-3 bioinks, in lessthan 20 s, while an industrial DMD-based printer would probably consumean additional time of 100 s simply devoted to manual bioink injectionsand switching. Other manually operated laboratory-scale multi-materialDMD-based printers would take similar time to replace the bioinks.Therefore, the bioprinter disclosed herein could achieve a speed fasterthan those of the existing stereolithography and/or DMD-based platforms.

The advantage of the bioprinting platform disclosed in the instantexample in terms of fabrication speed will become more noticeable whenfabrication time hampers cell viability in larger cell-laden constructs.The unique features of this bioprinter have significantly promoted thecurrent level of control and printing speed among existing bioprintingtechniques. This concept is also expandable to as many bioinks as neededby simply increasing the number of inlet channels. In addition, theprinting speed of our multi-material DMD bioprinting system may befurther improved by carefully coordinating projection light and localoxygen levels to achieve continuous photocrosslinking of the bioinks ina layerless manner.

Additional Experimental Procedures

DMD-Based Bioprinting Platform: FIG. 6 a shows the custom-builtDMD-based bioprinting system used for the fabrication of multi-componentconstructs. A UV LED (M385LP1-C1; Thorlabs, Newton, N.J.) mounted to alight collimator was used at a wavelength of 365 nm and a power ofnearly 500 mW/cm². A Newport (Nashua, N.H.) power meter was used todetermine the light intensity. Our digital models built with AutoCADwere converted to 2D bitmap slices and translated to spatially tilt apattern of micromirrors on the DMD panel (DLP® LightCrafter™ 6500; TexasInstruments Inc., Dallas, Tex.). A stage controller was used to managethe three-axis stage movement, whereas the UV light source (Thorlabs)was directed via an optical path towards the DMD panel at a specificangle to facilitate light reflection through the projection optics tothe stage. The thickness of the layers could be adjusted in 100-μm stepsand the planar resolution of the system was found to be around 10 μm(5×5 mm illumination area). This resolution was achieved utilizing acompound lens with 2-cm working distance (FIG. 6 b ). Since the DMDreflects an array of square pixels and the layers are built on top ofeach other, the tolerance of the printing is important in the resolutionof constructs [16]. Exposure times ranged from 1 to 20 s depending onthe aperture of light and hydrogel composition. The DMD panel, thestage, and the UV light source were controlled by a microcontroller(Arduino UNO; Arduino, Italy). FIG. 8 b shows the printing sequencecontrolled by the Arduino program. While the pattern is being printedthere is no liquid flowing in the chamber (stage 1 in FIG. 10 d ); oncethe pattern is printed, the motor brings the chip up (stage 2 in FIG. 10d ); if the material is to be changed, the valve of the next material isturned on and the new material starts washing the one currently in thechamber (stage 3 in FIG. 10 d ); an additional washing with buffer canbe added if the material is hard to wash; finally, the motor brings thechip down to print the following layer. An open chamber shown in FIG. 6c was also used to test single-material printing and characterize theoptical setup.

Optical configuration. The optical setup of the DMD-based bioprintingsystem (FIG. 6 b ) comprised of a UV lamp (M385LP1-C1; Thorlabs, Newton,N.J.), a DMD chipset (1050×920, DLP6500; Texas Instruments, Austin,Tex.), a Keplerian optical setup (Thorlabs), and a microscope objective(20×; Mitutoyo, Kanagawa, Japan). The optical beam path can be describedas follows: a uniform beam of light departing from the UV LED mounted tolight collimator is used at a wavelength of 385 nm and 1.650 mW. The UVbeam (f=57 mm) is reduced using a Keplerian optical setup (f=150 mm forUV-fused silica Plano convex lens and f=12.7 mm for UV bi-concave lens)with the aim of increasing the light intensity and cover full area ofthe DMD chipset (10 mm×12 mm). The resulting beam subsequently impingesupon the DMD chipset in which different light patterns, controlled usingCAD, can be generated. Using a microscope objective (Mitutuyo) theselected pattern from the DMD chipset was focused at a distancecorresponding to the focal length of the microscope objective (2 mm).This distance was considered the working distance where thephotosensitive hydrogel or polymer crosslinks can be exposed to UV lightto create the complex cell-laden constructs.

Hydrogel Preparations: PEGDA (Mn=700), 2,2,6,6-tetramethylpiperdine1-oxyl (TEMPO), and gelatin from porcine skin type A were purchased fromSigma-Aldrich (St. Louis, Mo.). TEMPO mitigates free radical migrationdistance leading to structure sharpness in high-aspect ratio constructs.High-purity distilled water was generated by Millipore system with aresistivity reading of 18.2 MO upon collection. Lithiumphenyl-2,4,6-trimethyl-benzoyl-phosphinate (LAP) was purchased fromBioBots (Philadelphia, Pa.). This photoinitiator has been extensivelyused for cellular studies. A 50% v/v PEGDA aqueous solution wasprepared, and TEMPO (0.01% w/v) and LAP (1.0% w/v) were added to thesolution. The mixture was heated to 80° C. for 1-3 h. The resultingPEGDA bioink was used to fabricate the structures presented in FIG. 7 .Food dyes and fluorescence beads were used to assist visualizations whennecessary. For 3D constructs and cell study we used GelMA-7% w/vsolution containing LAP (0.3% w/v), as shown in FIG. 12 . GeIMA was alsofabricated following well-established protocol. In summary, gelatin wasdissolved in PBS for 2 h at 60° C. under constant stirring to make a 10%w/v gelatin solution and then added 5% v/v methacrylic anhydride(Sigma-Aldrich), and subsequently stirred for 1 h at 60° C. and 500 rpm.Two volumes of pre-heated PBS were added and the solution was dialyzedfor one week.

Multi-Inlet Microfluidic Chip: The microfluidic chip of the bioprintingplatform (FIGS. 6 a and 8 a ) was designed and fabricated. The chipmodel was designed using CorelDraw (Corel Corp, Ottawa, ON) software andimported to a laser cutting machine (VLS 2.30 Desktop Laser, UniversalLaser Systems Inc, Richmond, Va.) for cutting PMMA sheets (1.6 mm inthickness; McMaster-Carr, Robbinsville, N.J.). The mold included four1-mm-wide inlets, connecting channels, one printing region of 10 mm indiameter, and one outlet. After merging the four inlets, the firstsemi-circular region was 4 mm wide and it connected to a short 2-mm-widechannel. A smooth connection to the printing region was designed withthree curved branches so that the hydraulic resistance of each branchwas approximately one-third the hydraulic resistance of one singlechannel. The four inlets were merged into a wider region (4 mm in width)with a total length of 5 mm. PDMS precursor (Sylgard 184; Dow Corning,Midland, Mich.), prepared by an elastomer/curing agent ratio of 10:1,was poured onto the PMMA mold, cured at 85° C. for 2 hr, and peeled off.A circular region assigned for printing was created using a customizedpunch (outer diameter: 10 mm) in the center of the PDMS chamber. Ourdesign also included a thin PDMS membrane; spin coating was used (at1000 rpm) of a PDMS drop on a glass slide, followed by curing at 85° C.for 2 hr, to achieve this membrane. The patterned PDMS replica wasmanually bonded to the membrane and then sandwiched between two PMMAsheets, as depicted in FIG. 8 a . To connect the inlets and outlet,stainless steel adaptors (outer diameter: 0.5 mm) were used.

Flow characterization of the microfluidic chip. A pressure-driven designwas chosen using a combination of compressed nitrogen gas, pneumaticvalves, syringe-based reservoirs, and Tygon medical tubing (FIG. 8 b ).Flow controllers (National Instruments, Woburn, Mass.) were connected toa nitrogen tank to provide the desired input pressure (0-25 psi).Standard 5-mL syringes were modified to be pushed by compressed nitrogenand attached to 20 G needles on the other side. We first characterizedflow mixing and washing using PEGDA-50% solutions with different colors(food dyes) and PBS for washing. FIG. 8 b-c shows the performance of thechip in terms of proper washing after each dye. To evaluate the floweffect on printed patterns and washing mechanism, a series of numericalsimulations were performed. To validate the simulation, a high-viscositygelatin solution (at 37° C.) was utilized to capture flow streamlines.The fluorescence micrographs at nine different fluidic path lengths foreach microchannel were acquired using a complementary metal oxidesemiconductor (CMOS) camera (Flea3.0; Point Grey, Vancouver, Canada). Inaddition, to evaluate the mixer performance, the residence time wascharacterized by injecting red, blue, and green bioinks at each inletand recording the color changes.

Computation fluid dynamic (CFD) simulation was used to demonstrate howfluid manipulation could enable washing non-crosslinked bioinks from thecrosslinked hydrogels. The CFD simulation was performed using a software(COMSOL Multiphysics; finite element scheme). The chip geometry wasimported from the CorelDraw software to COMSOL in DXF format. Theinitial observations (Video S1) confirmed that the flow of PEGDA waslaminar. A physics controlled triangular mesh was selected, for case oflaminar flow and physical properties of PEGDA-50% and PEGDA-10%(density: 1.12 g/cm3; viscosities: 30 to 1000 cP) were used for thecomputations. Assuming time-independent (i.e. steady-state) conditions,the flow was simulated for 4 s (40 time steps) to ensure proper washingof two-material simulation. The Reynolds number (0 to 10) confirmed thelaminar flow. Cartesian coordinates (Z out of plane) were selected todefine out computational model. The symmetry line Y=0, where thevertical velocity is zero, simplified our computations to half-geometry.The channel walls as well as the fluid-solid interface of patternedshapes (e.g. star) were all defined as no-slip boundary conditions. Theinput was defined as fluid velocity, while for the washing step wedefined the presence of fluid I in all channel and then fluid II at theinlet. Finally, for the case of 3D, a tetrahedral mesh was used alongwith fully coupled solver (and partially coupled) at extremely slow timerate (>1 hr per time step) for reasonable washing speed for laminar flow(FIG. 8 ).

From the above description, those skilled in the art will perceiveimprovements, changes and modifications. Such improvements, changes andmodifications are within the skill of one in the art and are intended tobe covered by the appended claims.

All patent applications, patents, and printed publications cited hereinare incorporated herein by reference in the entireties, except for anydefinitions, subject matter disclaimers or disavowals, and except to theextent that the incorporated material is inconsistent with the expressdisclosure herein, in which case the language in this disclosurecontrols.

What is claimed is:
 1. A printing system comprising a stereolithographicprinting device, a moving stage, and a microfluidic device; wherein themicrofluidic device comprises a plurality of reservoirs, each reservoirhousing a different ink for printing, and a microfluidic chip, whereinthe microfluidic chip comprises a chamber, wherein the chamber comprisesa plurality of inlets, a printing region, and one or more outlets, andwherein the stereolithographic printing device comprises a digitalmicromirror device.
 2. The printing system of claim 1, wherein thereservoirs are each connected to a respective inlet.
 3. The printingsystem of claim 1, wherein the microfluidic chip further comprises anelastic membrane.
 4. The printing system of claim 3, wherein the elasticmembrane comprises a polydimethylsiloxane (PDMS) polymer.
 5. Theprinting system of claim 3, wherein the elastic membrane is bonded tothe chamber.
 6. The printing system of claim 3, wherein the chamber iscomprised of PDMS polymer.
 7. The printing system of claim 5, whereinthe chamber bonded to the elastic membrane is sandwiched between twopolymer sheets.
 8. The printing system of claim 7, wherein the twopolymer sheets are comprised of poly(methyl methacrylate) (PMMA).
 9. Theprinting system of claim 1, wherein each reservoir is connected to apneumatic valve and wherein the pneumatic valves are each connected to acontainer comprising a gas.
 10. The printing system of claim 1, whereinthe inks comprise gelatin methacryloyl (GelMA) and poly(ethylene glycol)diacrylate (PEGDA).
 11. The printing system of claim 1, wherein the inkscomprise biologically active components, wherein the biological activecomponents comprise at least one of biomaterials, cells, growth factors,cytokines, anti-infection agents, adhesive molecules, and nanoparticles.12. The printing system of claim 1, wherein the chamber furthercomprises a mixer.